Our studies address a critical problem in hemoglobin (Hb) biology: how inherently unstable globin protein subunits are folded and maintained during normal and pathological erythropoiesis. We discovered alpha hemoglobin stabilizing protein (AHSP), an erythroid protein that specifically binds free ? globin subunit, stabilizes its structure and limits its pro-oxidant activities. Our preliminary studies suggest two distinct functions for AHSP. First, to detoxify excess ? globin that accumulates during normal erythropoiesis and in various anemias, particularly ? thalassemia. Second, to fold and stabilize newly formed ? globin subunits en route to HbA (?2??2) synthesis. Of potential importance to both functions, we discovered that degradation of AHSP mRNA is accelerated by iron, an essential component of HbA and determinant of nascent globin protein stability. Our overall view is that AHSP facilitates normal HbA synthesis and also buffers against imbalances that arise from genetic or environmental stresses, such as thalassemias and iron deficiency. Now we seek to better understand AHSP activities and their relevance to human health. Aim 1 uses mouse genetics to investigate AHSP functions in vivo. We will examine the consequences of manipulated AHSP expression in thalassemias and create Ahsp gene missense mutations in mice to probe mechanisms of AHSP protein function. Aim 2 studies the biochemical properties of AHSP. We will test in vitro if AHSP promotes reconstitution of HbA from its purified apo-globin and heme components and search for new erythroid proteins that interact with ? globin-AHSP complexes. Aim 3 examines the mechanisms by which iron regulates AHSP expression and the physiological implications of this pathway during altered iron homeostasis. If successful, our work will establish new basic principles of Hb biology and erythropoiesis. In addition, there are potential practical long-term benefits. For example, understanding how AHSP detoxifies excess ?Hb should illustrate novel therapeutic approaches for human ? thalassemias. Elucidating the role of AHSP in HbA synthesis may provide tools to optimize the manufacture of recombinant Hb-based blood substitutes. Finally, defining functional interactions between iron and AHSP could provide insights into the pathophysiology and management of iron overload and deficiency states. PUBLIC HEALTH RELEVANCE: Project Narrative: Our work examines how the blood oxygen carrier hemoglobin is stabilized and assembled during red blood cell formation. If successful, our experiments will enhance general knowledge about how blood is formed. In addition, we will provide new insights toward understanding and treating common and debilitating anemias such as thalassemia and iron deficiency. [unreadable] [unreadable] [unreadable]